LOCUS       sequence-340743-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PylB overexpression and purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6616)
  AUTHORS   Ho JML, Miller CA, Smith KA, Mattia JR, Bennett MR
  TITLE     Improved pyrrolysine biosynthesis through phage assisted 
            non-continuous directed evolution of the complete pathway.
  JOURNAL   Nat Commun. 2021 Jun 24;12(1):3914. doi: 10.1038/s41467-021-24183-9.
  PUBMED    34168131
REFERENCE   2  (bases 1 to 6616)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-06-24"; volume: "12(1):3914. doi"; pages: " 
            10.1038/s41467-021-24183-9"
COMMENT     Sequence Label: pJH767
FEATURES             Location/Qualifiers
     source          1..6616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1385..1576
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1570..1592)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1678..1817
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1733..1752
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1832..1849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2003..2591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2083..2102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2702..2721)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2713..3528
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2713..3528
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2786..2805)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3620..4075)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3757..3778)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3969..3988
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4112..4159)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4155..4173
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5268..5561)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(5577..5597)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5607..5624)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5643..5665
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5680..5704
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(5704..5723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5705..5723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5807..5826)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6032..6109
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6110..6616,1..576)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6129..6148)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
       61 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
      121 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
      181 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
      241 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
      301 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
      361 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
      421 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
      481 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      541 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
      601 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
      661 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
      721 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
      781 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
      841 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
      901 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
      961 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     1021 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     1081 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     1141 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     1201 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     1261 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     1321 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     1381 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     1441 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     1501 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     1561 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     1621 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     1681 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     1741 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     1801 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     1861 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     1921 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     1981 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2041 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2101 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2161 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2221 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2281 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2341 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2401 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     2461 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2521 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2581 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2641 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
     2701 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
     2761 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     2821 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     2881 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     2941 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     3001 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     3061 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     3121 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     3181 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     3241 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     3301 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     3361 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     3421 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     3481 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
     3541 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     3601 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3661 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3721 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3781 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3841 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3901 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3961 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4021 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     4081 tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc cctcaagacc cgtttagagg
     4141 ccccaagggg ttatgctagt tattgctcag cggtggcagc agccaactca gcttcctttc
     4201 gggctttgtt attagcagcg cagtacagtc tcgaattccg cctgtggcgc tggttccatg
     4261 cccattccct caaggcgctt gatgacgctt ttaatgtcgc ggtcgcgctc ttccaggtca
     4321 cgatcgtagt tagcaactcc ctcaagtttt gaatcactag gaaggatgct agtcacgata
     4381 ttagcacctg cgttaagacg gtgtaccatc ccgtcgatcc cttccaggtc caacgacgcg
     4441 gggattaagc acttcggaaa gatcagacgc aagatcgcaa tgattttcag ttcgctcagg
     4501 ttactatttt ccttaaaacg ctcaagggga gttccttcct ggggtaagaa agtcatcacg
     4561 cgaaccatgt cgggatcatt tgatttcatg ccgcgtaaag atttgatcgt tgactcgata
     4621 tcgttcccga ctcccgttaa gataccgtct tcaatgcaat acccttgttc tttagcaaaa
     4681 cgacgagtat tgtagcgccc cttgaacgac tgacctacgc gcaacttctc gtacaactct
     4741 tgatcatagg tttcttgata taaggcgaag aaattagctc ccttttcgcg tgcttttaaa
     4801 agggtagcat tatccattac tcctggcgaa atcatgattg gaagtccaag ctcattctta
     4861 accatttgga ccagctccac gaagcgatca ggttcctcgt aataataggg atcctccccc
     4921 atagtcagat caatcatatg gaaacccgca cctttaaggg ccttgcaagt ctctttgatt
     4981 tcttcgcggg acaaacgata gcggttaatc tcgtttttgc agttataata gcaaaacgca
     5041 cattggtttt tgcagtatgt gctgaagtag ataaaacagt tcaggaatac gcgatttcca
     5101 aagtaatggt tgcgtacttt acgtgcgacg taatacagct tttccaattc ttcttccgat
     5161 tccaggctaa gcaaagcgcg taaatcgtcg tcagataact gaaatccttc aataattttt
     5221 tcgccaaaac gatccagatc ttcagtcgcc atcttcttaa tcatatgacc accaatctgt
     5281 tctctgtgag cctcaataat atcgttatcc tccatgtcca aatcttcagg ggtctgatca
     5341 gcttgaattc taataccgtc gtacaagaat cttaaggagt ccatttcctt accctgtctt
     5401 ttagcgaacg cttccatcag ccttcttaaa ggagtggtct ttttgatctt gaagaagatc
     5461 tctgaagacc catcggacac ctttaaattg atgtgagtct caggcttgac ttctggcttg
     5521 acctctggct tagcttcttg attgacttct gagtccgaca tgctgctgcc catcatgccc
     5581 tgaaaataca ggttttcgcc gctgctgtga tgatgatgat gatggctgct gcccatggta
     5641 tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa
     5701 ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga
     5761 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     5821 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     5881 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     5941 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     6001 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     6061 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     6121 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     6181 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     6241 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     6301 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     6361 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     6421 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     6481 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     6541 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     6601 agacggtacg cgactg
//
